White matter tauopathy: Transient functional loss and novel myelin remodeling.

Early white matter (WM) changes are common in dementia and may contribute to functional decline. We here examine this phenomenon in an induced dementia model for the first time. We report a novel and selective form of myelin injury as the first manifestation of tauopathy in the adult central nervous system. Myelin pathology rapidly followed the induction of a P301 tau mutation associated with fronto-temporal dementia in humans (rTG4510 line). Damage involved focal disruption of the ad-axonal myelin lamella and internal oligodendrocyte tongue process, followed by myelin remodeling with features of re-myelination that included myelin thinning and internodal shortening. The evolution of the re-myelinated phenotype was complete in the molecular layer of the dentate gyrus after 1 month and in the optic nerve (ON) after 9 months of transgene induction and proceeded in the absence of actual demyelination, reactive glial changes or inflammatory response. The initial rapid myelin pathology was associated with loss of WM function and performance decline in a novel recognition test and both these effects largely reversed during the myelin re-modeling phase. The initial phase of myelin injury was accompanied by disruption of the vesicle population present in the axoplasm of hippocampal and ON axons. Axoplasmic vesicle release is significant for the regulation of myelin plasticity and disruption of this pathway may underlie the myelin damage and remodeling evoked by tauopathy. WM dysfunction early in tauopathy will disorder neural circuits, the current findings suggest this event may make a significant contribution to early clinical deficit in dementia.

The Leukocentric Theory of Neurological Disorder: A Manifesto.

Approximately half of the human brain is composed of white matter (WM), a specialized tissue housing the axonal projection of neurons and their necessary supporting glial cells. Axons course long distances from their parent soma, have a delicate structure, large surface area and in many cases are dependent upon a uniquely close morphological arrangement with myelinating oligodendrocyte partners; all factors that may predispose them to injury and disease. WM damage is central to a range of well-characterized disorders including multiple sclerosis and spinal cord injury and is also makes a significant contribution to disorders often considered to be largely focused in gray matter; for example, in stroke where ~49% of injury by volume is located in WM. In addition, advances in brain imaging have revealed early, often prodromal, changes in WM structure in most forms of neurodegeneration including Alzheimer's, Huntingdon's and Parkinson's diseases as well as during normal cognitive decline and a variety of behavioral conditions. The significance of the early WM changes for the etiology of these diseases is largely unknown. Subtle, early changes in synaptic function may produce the prodromal WM changes evident in imaging, or WM and gray mater structures may undergo simultaneous reactions to the underlying disease process. However, there are rational mechanisms for the transmission of pathology from WM to gray matter and this article suggests an alternative hypothesis: that WM pathology precedes and to some extent is causal of synaptic dysfunction in many common neurological disorders. Neurological disorders that have their origin or their principle lesion in WM are here defined as "leukopathologies".

HCO3(-)-independent pH regulation in astrocytes in situ is dominated by V-ATPase.

The mechanisms of HCO3(-)-independent intracellular pH (pHi) regulation were examined in fibrous astrocytes within isolated neonatal rat optic nerve (RON) and in cultured cortical astrocytes. In agreement with previous studies, resting pHi in cultured astrocytes was 6.82 ± 0.06 and inhibition of the V-ATPase H(+) pump by Cl(-) removal or via the selective inhibitor bafilomycin had only a small effect upon resting pHi and recovery following an acid load. In contrast, resting pHi in RON astrocytes was 7.10 ± 0.04, significantly less acidic than that in cultured cells (p < 0.001), and responded to inhibition of V-ATPase with profound acidification to the 6.3-6.5 range. Fluorescent immuno-staining and immuno-gold labeling confirmed the presence V-ATPase in the cell membrane of RON astrocyte processes and somata. Using ammonia pulse recovery, pHi recovery in RON astrocyte was achieved largely via V-ATPase with sodium-proton exchange (NHE) playing a minor role. The findings indicate that astrocytes in a whole-mount preparation such as the optic nerve rely to a greater degree upon V-ATPase for HCO3(-)-independent pHi regulation than do cultured astrocytes, with important functional consequences for the regulation of pH in the CNS.

Plasmalemmal and mitochondrial Na(+) -Ca(2+) exchange in neuroglia.

In the absence of the electrical signaling for which neurons are so highly specialized, GLIA rely on the slow propagation of ionic signals to mediate network events such as Ca(2+) and Na(+) waves. Glia differ from neurons in another important way, they are replete with a high density of ionic-transport proteins that are essential for them to fulfil their basic functions as guardians of the intra and extra-cellular milieux. Both the signaling and the homeostatic properties of glial cells are therefore particularly dependent upon the regulation of the two principle physiological metal cations, Ca(2+) and Na(+) . For both ions, glia express high-affinity/low capacity ATP-fuelled pumps that can rapidly move small numbers of ions against an electro-chemical gradient. For both Ca(2+) and Na(+) regulation, a single transporter family, the Na(+) -Ca(2+) exchanger (NCX), is used to maintain cellular ion homeostasis over the longer term and under conditions of prolonged or acute ionic dysregulation in astrocytes, oligodendroglia and microglia. Our understanding of glial NCX, both plasmalemmal and mitochondrial, is undergoing the kind of transformation that our understanding of glial cells, in general, has undergone in recent decades. These exchange proteins are becoming increasingly recognized for their essential roles in intracellular homeostasis while their signaling functions are starting to come to light. This review summarizes these key aspects and highlights the many areas where work has yet to begin in this rapidly evolving field. GLIA 2016;64:1646-1654.

White matter injury: Ischemic and nonischemic.

Ischemic pathologies of white matter (WM) include a large proportion of stroke and developmental lesions while multiple sclerosis (MS) is the archetype nonischemic pathology. Growing evidence suggests other important diseases including neurodegenerative and psychiatric disorders also involve a significant WM component. Axonal, oligodendroglial, and astroglial damage proceed via distinct mechanisms in ischemic WM and these mechanisms evolve dramatically with maturation. Axons may pass through four developmental stages where the pattern of membrane protein expression influences how the structure responds to ischemia; WM astrocytes pass through at least two and differ significantly in their ischemia tolerance from grey matter astrocytes; oligodendroglia pass through at least three, with the highly ischemia intolerant pre-oligodendrocyte (pre-Oli) stage linking the less sensitive precursor and mature phenotypes. Neurotransmitters play a central role in WM pathology at all ages. Glutamate excitotoxicity in WM has both necrotic and apoptotic components; the latter mediated by intracellular pathways which differ between receptor types. ATP excitotoxicity may be largely mediated by the P2X7 receptor and also has both necrotic and apoptotic components. Interplay between microglia and other cell types is a critical element in the injury process. A growing appreciation of the significance of WM injury for nonischemic neurological disorders is currently stimulating research into mechanisms; with curious similarities being found with those operating during ischemia. A good example is traumatic brain injury, where axonal pathology can proceed via almost identical pathways to those described during acute ischemia.

Neurotransmitter signaling in white matter.

White matter (WM) tracts are bundles of myelinated axons that provide for rapid communication throughout the CNS and integration in grey matter (GM). The main cells in myelinated tracts are oligodendrocytes and astrocytes, with small populations of microglia and oligodendrocyte precursor cells. The prominence of neurotransmitter signaling in WM, which largely exclude neuronal cell bodies, indicates it must have physiological functions other than neuron-to-neuron communication. A surprising aspect is the diversity of neurotransmitter signaling in WM, with evidence for glutamatergic, purinergic (ATP and adenosine), GABAergic, glycinergic, adrenergic, cholinergic, dopaminergic and serotonergic signaling, acting via a wide range of ionotropic and metabotropic receptors. Both axons and glia are potential sources of neurotransmitters and may express the respective receptors. The physiological functions of neurotransmitter signaling in WM are subject to debate, but glutamate and ATP-mediated signaling have been shown to evoke Ca(2+) signals in glia and modulate axonal conduction. Experimental findings support a model of neurotransmitters being released from axons during action potential propagation acting on glial receptors to regulate the homeostatic functions of astrocytes and myelination by oligodendrocytes. Astrocytes also release neurotransmitters, which act on axonal receptors to strengthen action potential propagation, maintaining signaling along potentially long axon tracts. The co-existence of multiple neurotransmitters in WM tracts suggests they may have diverse functions that are important for information processing. Furthermore, the neurotransmitter signaling phenomena described in WM most likely apply to myelinated axons of the cerebral cortex and GM areas, where they are doubtless important for higher cognitive function.

Central axons preparing to myelinate are highly sensitive [corrected] to ischemic injury.

OBJECTIVE: Developing central white matter is subject to ischemic-type injury during the period that precedes myelination. At this stage in maturation, central axons initiate a program of radial expansion and ion channel redistribution. Here we test the hypothesis that during radial expansion axons display heightened ischemic sensitivity, when clusters of Ca(2+) channels decorate future node of Ranvier sites. METHODS: Functionality and morphology of central axons and glia were examined during and after a period of modeled ischemia. Pathological changes in axons undergoing radial expansion were probed using electrophysiological, quantitative ultrastructural, and morphometric analysis in neonatal rodent optic nerve and periventricular white matter axons studied under modeled ischemia in vitro or after hypoxia-ischemia in vivo. RESULTS: Acute ischemic injury of central axons undergoing initial radial expansion was mediated by Ca(2+) influx through Ca(2+) channels expressed in axolemma clusters. This form of injury operated only in this axon population, which was more sensitive to injury than neighboring myelinated axons, smaller axons yet to initiate radial expansion, astrocytes, or oligodendroglia. A pharmacological strategy designed to protect both small and large diameter premyelinated axons proved 100% protective against acute ischemia studied under modeled ischemia in vitro or after hypoxia-ischemia in vivo. INTERPRETATION: Recent clinical data highlight the importance of axon pathology in developing white matter injury. The elevated susceptibility of early maturing axons to ischemic injury described here may significantly contribute to selective white matter pathology and places these axons alongside preoligodendrocytes as a potential primary target of both injury and therapeutics.

Identification of four functional NR3B isoforms in developing white matter reveals unexpected diversity among glutamate receptors.

Functional neurotransmitter receptors are expressed in central white matter, where they mediate ischemic damage to glia and may be involved in cell-cell signalling. In this study, we analysed NMDA receptor NR1, NR2B-C and NR3A-B subunit expression in the brain and optic nerve by molecular cloning. In addition to the canonical forms of NR1 and NR2, four previously unknown NR3B variants, generated by alternative splicing, were identified. The variants encoded for isoforms with deletions of 8/15 amino acids in the N-terminal domain or 200/375 amino acids removing one or three transmembrane domains and part of the C-terminal domain, as compared with the previously characterized NR3B isoform. Co-expression of NR3B isoforms with NR1/NR2A-C modulated the amplitude and Mg(2+)-sensitivity of glutamate responses in a NR2 subunit-dependent fashion, with significant variations in the effects produced by different isoforms. These effects were not the result of reduced surface expression of the receptor complex since all NR3B isoforms reduced surface expression by a similar degree. These data reveal previously uncharacterized regulation of NMDA receptor function by alternative splicing of the NR3B subunit.

Novel morphological features of developing white matter pericytes and rapid scavenging of reactive oxygen species in the neighbouring endothelia.

Capillary endothelia and pericytes form a close morphological arrangement allowing pericytes to regulate capillary blood flow, in addition to contributing to vascular development and support. Vascular changes associated with oxidative stress are implicated in important pathologies in developing whiter matter, but little is known about the vascular unit in white matter of the appropriate age or how it responds to oxidative stress. We show that the ultrastructural arrangement of post-natal day 10 (P10) capillaries involves the apposition of pericyte somata to the capillary inner basement membrane and penetration of pericyte processes onto the abluminal surface where they form close connections with endothelial cells. Some pericytes have an unusual stellate morphology, extending processes radially from the vessel. Reactive oxygen species (ROS) were monitored with the ROS-sensitive dye 2',7'-dichlorofluorescin (DCF) in the endothelial cells. Exposure to exogenous ROS (100 µm H(2) O(2) or xanthine/xanthine oxidase), evoked an elevation in intracellular ROS that declined to baseline during the ongoing challenge. A second challenge failed to evoke an intracellular ROS rise unless the nerve was rested for > 4 h or exposed to very high levels of exogenous ROS. Exposure to a first ROS challenge prior to loading with DCF also prevented the intracellular ROS rise from a second challenge, proving that dye washout during exposure to ROS is not responsible for the loss of a second response. Perfusion with 30 µm extracellular Ca(2+) or the voltage-gated Ca(2+) antagonist diltiazem partially prevented this rapid scavenging of intracellular ROS, but blocking either catalase or glutathione peroxidase did not. The phenomenon was present over a range of post-natal ages and may contribute to the high ROS-tolerance of endothelial cells and act to limit the release of harmful ROS onto neighbouring pericytes.

NMDA receptors are expressed in developing oligodendrocyte processes and mediate injury.

Injury to oligodendrocyte processes, the structures responsible for myelination, is implicated in many forms of brain disorder. Here we show NMDA (N-methyl-D-aspartate) receptor subunit expression on oligodendrocyte processes, and the presence of NMDA receptor subunit messenger RNA in isolated white matter. NR1, NR2A, NR2B, NR2C, NR2D and NR3A subunits showed clustered expression in cell processes, but NR3B was absent. During modelled ischaemia, NMDA receptor activation resulted in rapid Ca2+-dependent detachment and disintegration of oligodendroglial processes in the white matter of mice expressing green fluorescent protein (GFP) specifically in oligodendrocytes (CNP-GFP mice). This effect occurred at mouse ages corresponding to both the initiation and the conclusion of myelination. NR1 subunits were found mainly in oligodendrocyte processes, whereas AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptor subunits were mainly found in the somata. Consistent with this observation, injury to the somata was prevented by blocking AMPA/kainate receptors, and preventing injury to oligodendroglial processes required the blocking of NMDA receptors. The presence of NMDA receptors in oligodendrocyte processes explains why previous studies that have focused on the somata have not detected a role for NMDA receptors in oligodendrocyte injury. These NMDA receptors bestow a high sensitivity to acute injury and represent an important new target for drug development in a variety of brain disorders.

Glutamate receptor-mediated ischemic injury of premyelinated central axons.

OBJECTIVE: Ischemic injury of axons is a feature of periventricular leukomalacia, a pathological correlate of cerebral palsy. Recent evidence suggests that axons are damaged before they receive the first layer of compact myelin. Here we examine the cellular mechanisms underlying ischemic-type injury of premyelinated central axons. METHODS: Two-thirds of axons in the postnatal day 10 (P10) rat optic nerve are small premyelinated axons (<0.4microm in diameter), and one-third have undergone radial expansion in preparation for glial contact and the onset of myelination. Compound action potential recording and quantitative electron microscopy were used to examine the effect of modeled ischemia (oxygen-glucose deprivation) upon these two axon populations. Glutamate receptor (GluR) expression was investigated using polymerase chain reaction (PCR) and immunostaining approaches at the confocal light and ultrastructural levels. RESULTS: Oxygen-glucose deprivation produced action potential failure and focal breakdown of the axolemma of small premyelinated axons at sites of contact with oligodendrocyte processes, which were also disrupted. The resulting axon loss was Ca(2+)-dependent, Na(+)- and Cl(-)-independent, and required activation of N-methyl-D-aspartic acid (NMDA) and non-NMDA GluRs. NMDA receptor expression was localized to oligodendrocyte processes at sites of contact with premyelinated axons, in addition to expression within compact myelin. No periaxonal NMDA receptor expression was observed on oligodendrocyte processes ensheathing large premyelinated axons and no protective effect of GluR block was observed in these axons. INTERPRETATION: NMDA receptor-mediated injury to oligodendrocyte processes navigating along small premyelinated axons precedes damage to the underlying axon, a phenomena that is lost following radial expansion and subsequent oligodendrocyte ensheathment.

Conduction block and glial injury induced in developing central white matter by glycine, GABA, noradrenalin, or nicotine, studied in isolated neonatal rat optic nerve.

The damaging effects of excessive glutamate receptor activation have been highlighted recently during injury in developing central white matter. We have examined the effects of acute exposure to four other neurotransmitters that have known actions on white matter. Eighty minutes of Glycine or GABA-A receptor activation produced a significant fall in the compound action potential recorded from isolated post-natal day 10 rat optic nerve. This effect was largely reversed upon washout. Nicotinic acetylcholine receptor (nAChR) or adrenoreceptor activation with noradrenalin resulted in an approximately 35% block of the action potential that did not reverse during a 30-min washout period. While the effect of nAChR activation was blocked by a nAChR antagonist, the effect of noradrenalin was not ablated by alpha- or beta-adrenoreceptor blockers applied alone or in combination. In the absence of noradrenalin, co-perfusion with alpha- and beta-adrenoreceptor blockers resulted in nonreversible nerve failure indicating that tonic adrenoreceptor activation is required for nerve viability, while overactivation of these receptors is also damaging. Nerves exposed to nAChR + adrenoreceptor activation showed no axon pathology but had extensive glial injury revealed by ultrastructural analysis. Oligodendroglia exhibited regions of membrane vacuolization while profound changes were evident in astrocytes and included the presence of swollen and expanded mitochondria, vacuolization, cell processes disintegration, and membrane breakdown. Blinded assessment revealed higher levels of astrocyte injury than oligodendroglial injury. The findings show that overactivation of neurotransmitter receptors other than those for glutamate can produce extensive injury to developing white matter, a phenomenon that may be clinically significant.

Glutamate receptors and white matter stroke.

White matter (WM) damage during ischemia occurs at multiple sites including myelin, oligodendrocytes, astrocytes and axons. A major driver of WM demise is excitoxicity as a consequence of excessive glutamate release by vesicular and non-vesicular mechanisms from axons and glial cells. This results in over-activation of ionotropic glutamate receptors (GluRs) profusely expressed by all cell compartments in WM. Thus, blocking excitotoxicity in WM with selective antagonists of those receptors has a potential therapeutic value. The significance of WM GluR expression for WM stroke injury is the focus of this review, and we will examine the role of GluRs in injury to myelin, oligodendrocytes, astrocytes and the axon cylinder.

Vesicular apparatus, including functional calcium channels, are present in developing rodent optic nerve axons and are required for normal node of Ranvier formation.

P/Q-type calcium channels are known to form clusters at the presynaptic membrane where they mediate calcium influx, triggering vesicle fusion. We now report functional P/Q channel clusters in the axolemma of developing central axons that are also associated with sites of vesicle fusion. These channels were activated by axonal action potentials and the resulting calcium influx is well suited to mediate formation of a synaptic style SNARE complex involving SNAP-25, that we show to be located on the axolemma. Vesicular elements within axons were found to be the sole repository of vesicular glutamate in developing white matter. The axonal vesicular elements expressed the glutamate transporter V-ATPase, which is responsible for vesicular glutamate loading. The P/Q channel alpha(1A) subunit was found to be present within the axolemma at early nodes of Ranvier and deleterious mutations of the alpha(1A) subunit, or an associated alpha(2)delta-2 subunit, disrupted the localization of nodal proteins such as voltage-gated sodium channels, beta IV spectrin and CASPR-1. This was associated with the presence of malformed nodes of Ranvier characterized by an accumulation of axoplasmic vesicles under the nodal membrane. The data are consistent with the presence of a vesicular signalling pathway between axons and glial cells that is essential for proper development of the node of Ranvier.

Functional glutamate transport in rodent optic nerve axons and glia.

Recent findings suggest that synaptic-type glutamate signaling operates between axons and their supporting glial cells. Glutamate reuptake will be a necessary component of such a system. Evidence for glutamate-mediated damage of oligodendroglia somata and processes in white matter suggests that glutamate regulation in white matter structures is also of clinical importance. The expression of glutamate transporters was examined in postnatal Day 14-17 (P14-17) mouse and in mature mouse and rat optic nerve using immuno-histochemistry and immuno-electron microscopy. EAAC1 was the major glutamate transporter detected in oligodendroglia cell membranes in both developing and mature optic nerve, while GLT-1 was the most heavily expressed transporter in the membranes of astrocytes. Both EAAC1 and GLAST were also seen in adult astrocytes, but there was little membrane expression of either at P14-17. GLAST, EAAC1, and GLT-1 were expressed in P14-17 axons with marked GLT-1 expression in the axolemma, while in mature axons EAAC1 was abundant at the node of Ranvier. Functional glutamate transport was probed in P14-17 mouse optic nerve revealing Na+-dependent, TBOA-blockable uptake of D-aspartate in astrocytes, axons, and oligodendrocytes. The data show that in addition to oligodendroglia and astrocytes, axons represent a potential source for extracellular glutamate in white matter during ischaemic conditions, and have the capacity for Na(+)-dependent glutamate uptake. The findings support the possibility of functional synaptic-type glutamate release from central axons, an event that will require axonal glutamate reuptake.

The mechanisms of acute ischemic injury in the cell processes of developing white matter astrocytes.

Astrocytes are fundamentally important to the maintenance and proper functioning of the central nervous system. During the period of development when myelination is occurring, white matter astrocytes are particularly sensitive to ischemic injury and their failure to regulate glutamate during ischemic conditions may be an important factor in excitotoxic injury. Here, we have identified key mechanisms of injury that operate on the processes of immature white matter astrocytes during oxygen-glucose deprivation (OGD) using GFAP-GFP mice. Oxygen-glucose deprivation produced a parallel loss of astrocyte processes and somata, assessed by both the retention of GFP fluorescence within these structures and by quantitative electron microscopy. Oxygen-glucose deprivation-induced process loss was Ca(2+) independent and had two distinct mechanisms. Substituting either extracellular Na(+) or Cl(-), or perfusion with the Na-K-Cl co-transport blocker bumetanide, provided protection up to 40 mins of OGD but not beyond that point. HCO(3)(-) substitution or perfusion with 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid provided complete protection of the processes up to 60 mins of OGD. Zero-Na(+)/zero-K(+) conditions provided complete protection from OGD-induced injury of processes and somata at all time points. We conclude that acute ischemic-type injury of immature astrocytes follows a cytotoxic ion influx mediated in part by Na-K-Cl co-transport and in part by Na(+)- and K(+)-dependent HCO(3)(-) transport, a mechanism that is common to both cell processes and somata. This work provides a basis on which preventative strategies may be developed to protect white matter astrocytes from ischemic injury in susceptible individuals.

Vesicular glutamate release from central axons contributes to myelin damage.

The axon myelin sheath is prone to injury associated with N-methyl-D-aspartate (NMDA)-type glutamate receptor activation but the source of glutamate in this context is unknown. Myelin damage results in permanent action potential loss and severe functional deficit in the white matter of the CNS, for example in ischemic stroke. Here, we show that in rats and mice, ischemic conditions trigger activation of myelinic NMDA receptors incorporating GluN2C/D subunits following release of axonal vesicular glutamate into the peri-axonal space under the myelin sheath. Glial sources of glutamate such as reverse transport did not contribute significantly to this phenomenon. We demonstrate selective myelin uptake and retention of a GluN2C/D NMDA receptor negative allosteric modulator that shields myelin from ischemic injury. The findings potentially support a rational approach toward a low-impact prophylactic therapy to protect patients at risk of stroke and other forms of excitotoxic injury.

Hypoxia-ischemia preferentially triggers glutamate depletion from oligodendroglia and axons in perinatal cerebral white matter.

Ischemia is implicated in periventricular white matter injury (PWMI), a lesion associated with cerebral palsy. PWMI features selective damage to early cells of the oligodendrocyte lineage, a phenomenon associated with glutamate receptor activation. We have investigated the distribution of glutamate in rat periventricular white matter at post-natal day 7. Immuno-electron microcopy was used to identify O4(+) oligodendroglia in control rats, and a similar approach was employed to stain glutamate in these cells before and after 90 mins of hypoxia-ischemia. This relatively brief period of hypoxia-ischemia produced mild cell injury, corresponding to the early stages of PWMI. Glutamate-like reactivity was higher in oligodendrocytes than in other cell types (2.13+/-0.25 counts/microm(2)), and declined significantly during hypoxia-ischemia (0.93+/-0.15 counts/microm(2): P<0.001). Astrocytes had lower glutamate levels (0.7+/-0.07 counts/microm(2)), and showed a relatively small decline during hypoxia-ischemia. Axonal regions contained high levels of glutamate (1.84+/-0.20 counts/microm(2)), much of which was lost during hypoxia-ischemia (0.72+/-0.20 counts/microm(2): P>0.001). These findings suggest that oligodendroglia and axons are the major source of extracellular glutamate in developing white matter during hypoxia-ischemia, and that astrocytes fail to accumulate the glutamate lost from these sources. We also examined glutamate levels in the choroid plexus. Control glutamate levels were high in both choroid epithelial (1.90+/-0.20 counts/microm(2)), and ependymal cells (2.20+/-0.28 counts/microm(2)), and hypoxia-ischemia produced a large fall in ependymal glutamate (0.97+/-0.08 counts/microm(2): P>0.001). The ependymal cells were damaged by the insult and represent a further potential source of glutamate during ischemia.

An alternative surgical approach reduces variability following filament induction of experimental stroke in mice.

Animal models are essential for understanding the pathology of stroke and investigating potential treatments. However, in vivo stroke models are associated, particularly in mice, with high variability in lesion volume. We investigated whether a surgical refinement where reperfusion is not reliant on the Circle of Willis reduced outcome variability. Mice underwent 60 min of transient middle cerebral artery occlusion avoiding ligation of the external carotid artery. During reperfusion, the common carotid artery was either ligated (standard approach), or it was repaired to allow re-establishment of blood flow through the common carotid artery. All mice underwent MRI scanning for assessment of infarct volume, apparent diffusion coefficient and fractional anisotropy, along with terminal assessment of infarct volume by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Repairing the common carotid artery following middle cerebral artery occlusion enhanced reperfusion (P<0.01) and reduced the variability seen in both total (histological analysis, P=0.008; T2-weighted MRI, P=0.015) and core (diffusion tensor MRI, P=0.043) lesion volume. Avoiding external carotid artery ligation may improve animal wellbeing, through reduced weight loss, while using an alternative surgical approach that enabled reperfusion through the common carotid artery decreased the variability in lesion volume seen within groups.